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As the spread of antibiotic resistance threatens our ability to treat infections, avoiding the return of a pre-antibiotic era urgently requires the discovery of novel antibiotics. Actinomycetes, a family of bacteria commonly isolated from soil, are a proven source of clinically useful antibiotics. However, easily identifiable metabolites have been exhausted and the rediscovery of common antibiotics thwarts searches for rarer molecules. Sequencing of actinomycete genomes reveals that they contain far more biosynthetic gene clusters with the potential to encode antibiotics than whose products can be readily observed in the laboratory. The work presented in this thesis revolves around developing approaches to mine these previously inaccessible metabolites as a source of new antibiotics. First, I describe how inactivation of biosynthetic gene clusters for common antibiotics can uncover rare antibiotics otherwise masked in these strains. By applying CRISPR-Cas9 to knockout genes encoding nuisance antibiotics, I develop a simple strategy to reveal the hidden biosynthetic potential of actinomycete strains that can be used to discover rare or novel antibiotics. Second, I describe the use of the evolutionary history of biosynthetic gene clusters to prioritize divergent members of an antibiotic family, the glycopeptide antibiotics, that are likely to possess new biological activities. Using these predictions, I uncover a novel functional class of glycopeptide antibiotics that blocks the action of autolysins, essential peptidoglycan hydrolases required for remodelling the cell wall during growth. Finally, I apply target-directed genome mining, which makes use of target duplication as a predicted resistance mechanism within an antibiotic’s biosynthetic gene cluster. Using this approach, I discover the association of a family of gene clusters with the housekeeping protease ClpP and characterize the produced metabolite’s effect on ClpP function. These three research projects mine previously inaccessible chemical matter from a proven source of antibiotics, actinomycetes. The techniques and antibiotics described are required now more than ever to develop life-saving antibiotics capable of combatting multidrug-resistant pathogens. / Dissertation / Doctor of Philosophy (PhD) / Antibiotics are essential for treating life-threatening infections, but the rise of antibiotic resistance renders them ineffective. To treat these drug-resistant infections, new antibiotics that work in new ways are required. A family of bacteria commonly isolated from soil called Actinomycetes produce most antibiotics we use today, but it has become increasingly difficult to find new antibiotics from this source. My work describes three techniques that can be applied to actinomycetes to help overcome the challenges associated with antibiotic discovery. Specifically, these techniques guide discovery efforts by making use of regions in actinomycete genomes called biosynthetic gene clusters that often encode antibiotics. In doing so, I describe ways to uncover rare antibiotics from actinomycete strains that produce common and uninteresting antibiotics, use antibiotic family trees to discover antibiotics that work in new ways, and apply antibiotic resistance to identify biosynthetic gene clusters likely to act on a certain bacterial target.

<div>Cyclic peptides are important sources of medicines. </div><div>They are advantageous compared to linear peptides because they possess lower flexibility, which allows for high-affinity target binding and enhanced proteolytic stability. Unfortunately, achieving head-to-tail cyclization of peptides is quite challenging, as it is hard to control efficiency and regiospecificity of peptide macrocyclization. Many have attempted to improve peptide cyclization, including the use of different synthetic reagents as well as synthetic techniques to allow amide-bond formation and promote cyclization. While these strategies have offered great potential solutions, the aim of this study is to explore an alternative strategy that utilizes biocatalysis as a method of achieving successful peptide cyclization. Biocatalysis is the use of enzymes as natural process catalysts under artificial in vitro conditions. Biocatalysis is often more environmentally friendly and safer compared to traditional organic synthesis methods. Non-ribosomal peptide synthetases (NRPSs) are one of the major sources of cyclic peptides in nature. These are systems of large multifunctional proteins are organized into functional domains that act as an assembly line to generate peptide natural products. Normally, the thioesterase domain is responsible for hydrolysis and cyclization of the peptide. Recently, a novel cyclase (SurE) that is physically discrete from the NRPS was discovered. Based on this unique quality, we hypothesized that SurE would be easier to express compared to thioesterase domains and, for this reason, SurE could be a fantastic biocatalyst for the cyclization of peptides. To test this, we designed and generated an expression vector for SurE. We then expressed and purified the SurE protein. We also synthesized three linear peptides of varying lengths. To test for SurE activity, we attempted to add N-acetylcysteamine (SNAC) to mimic its native substrate. Unfortunately, we were unable to successfully attach the SNAC to our linear peptide. To combat this issue, a new synthesis strategy is currently being developed. This work is currently ongoing in the Parkinson lab, with the aim being to test the SurE protein, as well as other PBP-like cyclases, on other modified linear peptides and demonstrate whether the protein has the ability to cyclase a wide scope of peptides.</div><div><br></div>

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Several lipopeptidyl nucleoside antibiotics that inhibit bacterial translocase I (MraY) involved in peptidoglycan cell wall biosynthesis contain an aminoribosyl moiety, an unusual sugar appendage in natural products. A-90289 and muraminomicin are the two representative antibiotics that belong to this family. Bioinformatic analysis of the biosynthetic A-90289 gene clusters revealed that five enzymes are likely involved in the assembly and attachment of the aminoribosyl unit. These enzymes of A-90289 are functionally assigned by in vitro characterization. The results reveal a unique ribosylation pathway that highlighted by uridine-5′-monophosphate as the source of the sugar, a phosphorylase strategy to generate a sugar-1-phosphate, and a primary amine-requiring nucleotidylyltransferase that generates the NDP-sugar donor. Muraminomicin, which has a structure similar to A-90289, holds the distinction in that both ribose units are 2-deoxy sugars. The biosynthetic gene cluster of muraminomicin has been identified, cloned and sequenced, and bioinformatic analysis revealed a minimum of 24 open reading frames putatively involved in the biosynthesis, resistance, and regulation of muraminomicin. Similar to the A-90289 pathway, fives enzymes are still likely involved in the assembly of the 2,5-dideoxy-5-aminoribose saccharide unit, and two are now functionally assigned and characterized: Mra20, a 5′-amino-2′,5′-dideoxyuridine phosphorylase and Mra23, a UTP:5-amino-2,5-dideoxy-α-D-ribose-1-phosphate uridylyltransferase. The cumulative results are consistent with the incorporation of the ribosyl appendage of muraminomicin via the archetypical sugar biosynthetic pathway that parallels A-90289 biosynthesis